Among the molecular mechanisms that have been proposed to contribute to long-term potentiation in hippocampus are the activation and autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Here we report that high, but not low frequency stimulation applied to two groups of CA1 afferents resulted in a long lasting increase in the Ca(2+)-independent and total activities of the enzyme as well as an increase in the ratio of Ca(2+)-independent to total activity. The effect was obtained using two different CaM kinase II substrates, it was observed in hippocampal slices and in hippocampal organotypic cultures, and it could be blocked by preincubation of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoate. Treatment of slices with calyculin A, a phosphatase inhibitor, modified the activity of the enzyme, but long term potentiation could still be induced and a further increase in Ca(2+)-independent CaM kinase II activity still observed.